The genome of an organism is unique. Not only do the genomes of different species differ, but the genomes of different individuals within a species differ (with the exception of identical twins or clones). These differences provide individual and species specific characteristics which can be used for identification by nucleic acid biochemical techniques such as hybridization and polymerase mediated reactions, both dependent for their specificity on precise base pairing.
The goal of nucleic acid based diagnostics is the detection of specific nucleic acid sequences. This goal often requires the detection of a specific sequence in the presence of other sequences. In certain cases it is necessary to discriminate between closely related sequences, even sequences which differ by only a single nucleotide. Prior art methods for doing so are described in various publications. For example, the use of allele-specific oligonucleotide (ASO) hybridization probes for the detection of specific nucleic acid sequences has been described (Wu et al., DNA 8:135-142 (1989); Thein, et al., Br. J. Haematol. 70:225-231 (1988); Connor, et al., Proc. Natl. Acad. Sci. USA 80:278-282 (1983); Studencki, et al., Am. J. Hum. Genet. 37:42-51 (1985); Pirastu, et al., N.Engl.J.Med. 309:284-287 (1983); Orkin, et al., J.Clin.Invest. 71:775-779 (1983); Thein and Wallace, The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders. In Human genetic diseases: A practical approach. K. E. Davies, ed. (Oxford; IRL Press), pp. 33-50 (1986)). This approach allows the discrimination between nucleic acids which differ by as little as a single nucleotide (e.g., alleles). Individual hybridization reactions are required for each allele to be detected. Erlich, et al., Eur.J.Immunogenet. 18:33-55 (1991) and Zhang, et al., Nucleic Acids Res. 19:3929-3933 (1991) have recently described the use of immobilized ASO probes. In this method, a set of ASO probes is immobilized on a membrane and hybridized with labeled polymerase chain reaction (PCR) products. Under appropriate conditions hybridization is allele specific. Each hybridization can analyze only a single amplification reaction. The present invention allows for the detection of specific sequences in a sample. Because the template specific step and the detection step are each controlled by specific but independent base pairing requirements, the overall process allows detection of multiple templates and multiple samples simultaneously.
The concept of in vitro DNA amplification was first proposed by Khorana and coworkers in 1971 (Kleppe, et al., J.Mol.Biol. 56:341-361 (1971)). Realizing that total chemical synthesis of a gene would result in a finite amount of product, a procedure for in vitro replication was proposed. Their procedure was based on extensive studies of the repair replication reaction, the in vitro replication of a DNA template using a complementary primer (Kleppe, supra). Their proposal was as follows: "The DNA duplex would be denatured to form single strands. This denaturation step would be carried out in the presence of a sufficiently large excess of the two appropriate primers. Upon cooling, one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme." More recently, this in vitro amplification process has been further developed into the polymerase chain reaction (Mullis, et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Saiki, et al., Science 230:1350-1354 (1985); U.S. Pat. No. 4,683,202). Although template amplification improves detection of a particular sequence because a larger amount of template is available for analysis, post amplification steps are often required to detect specific sequences within the amplified product. For example, ASO hybridization has been combined with PCR amplification for the specific detection of various disease alleles (Impraim, et al., Biochem. Biophys. Res. Commun. 142:710-716 (1987); Saiki, et al., Nature 324:163-166 (1986); Farr, et al., Proc.Natl. Acad.Sci. USA 85:1629-1633 (1988); Saiki, et al., N.Engl.J.Med. 319:537-541 (1988); Chehab, et al., Nature 329:293-294 (1987)). The present invention provides an alternative for the analysis of PCR amplification products to determine the presence or absence of specific sequences.